CN104829707B - 一条cd环和e螺旋区突变的瘦素活性肽及其编码基因和应用 - Google Patents
一条cd环和e螺旋区突变的瘦素活性肽及其编码基因和应用 Download PDFInfo
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Abstract
本发明公开了一种CD环和E螺旋区突变的瘦素活性肽及其编码基因和应用。CD环和E螺旋区突变的瘦素活性肽的氨基酸序列如SEQ ID NO.1所示。本发明对人源Leptin的氨基酸序列进行修饰，以提高其脂肪降解的效率。利用化学方法分别合成CD环和E螺旋区突变的瘦素活性肽，尽可能的降低蛋白质空间结构对其活性的影响，从而提高对脂肪细胞的靶向性和降解作用，实际结果表明，本发明的CD环和E螺旋区突变的瘦素活性肽的脂肪细胞降解活性明显优于阳性对照‑人源瘦素蛋白和市售药物罗格列酮，在脂肪细胞分化前期、分化期和成熟脂肪细胞期三个时期均具有明显的细胞裂解作用，并且效果显著优于H‑LEP和罗格列酮，从而为研制新型、高效、廉价的减肥和降血糖药物提供候选靶标。
Description
技术领域：
本发明属于生物化学与分子生物学领域，具体涉及一条CD环和E螺旋区突变的瘦素活性肽及其编码基因和应用。
背景技术：
肥胖是人类健康最大的敌人之一，既是一个独立的疾病，又是Ⅱ型糖尿病、心血管病、高血压、脑中风和多种癌症的致病诱因，被世界卫生组织列为导致疾病负担的十大威胁之一，是不容忽视的全球性的问题。据报道，全世界每年约有1800万人死于由糖尿病和高血压引发的心脑血管疾病，而肥胖是糖尿病和高血压的关键致病因素。肥胖症严重危害了人类的健康，给社会造成巨大的损失。据报道2003年，我国因为肥胖、糖尿病等慢性疾病的支出就达到1.2万亿元，占GDP的10.3％。增长速度已经高于国民生产总值的增长速度。目前国内减肥市场需求十分广阔，2010年减肥品消费额达600亿元，而与肥胖症有直接或间接关系的疾病如糖尿病、心血管疾病等所造成的损失更是无法估计。目前用于肥胖症和糖尿病治疗的药物-非诺贝特和罗格列酮，虽然能够起到降低血脂和血糖的作用，但是长期服用会对人体造成严重的副作用，如胰岛素耐受和三致作用等，因此临床上肥胖症和糖尿病的治疗急需安全、高效、廉价的新型药物。
瘦素(Leptin)是由脂肪组织分泌的激素类物质，通过六种Leptin受体参与机体的脂肪代谢和食欲调节。本世际初期，瘦素被发现并且在肥胖老鼠模型上试验获得成功，引起科学界的轰动，预示着生物技术为全球数亿的肥胖患者找到了减肥新药。1999年科学家研究发现，人源瘦素蛋白的第116－130个氨基酸组织的多肽片段具有明显的脂肪降解活性。随后，美国科学家率先进行第一次瘦素蛋白人体实验，然而在73个受试的肥胖患者中，大部分患者的减肥效果不明显，只有2名患者在24周内减去35磅，这说明用野生型人源瘦素蛋白很难得到理想的减肥效果。
发明内容：
本发明的第一个目的是提供一种具有非常好的脂肪细胞降解活性的CD环和E螺旋区突变的瘦素活性肽。
本发明的CD环和E螺旋区突变的瘦素活性肽，其氨基酸序列如SEQ ID NO.1所示。
本发明的第二个目的是提供一种上述CD环和E螺旋区突变的瘦素活性肽的编码基因。
本发明的第三个目的是提供上述CD环和E螺旋区突变的瘦素活性肽在制备降解脂肪细胞药物、减肥药物或降血糖药物中的应用。
本发明对人源Leptin的氨基酸序列进行修饰，以提高其脂肪降解的效率。利用化学方法分别合成CD环和E螺旋区突变的瘦素活性肽，尽可能的降低蛋白质空间结构对其活性的影响，从而提高对脂肪细胞的靶向性和降解作用，实际结果表明，本发明的CD环和E螺旋区突变的瘦素活性肽的脂肪细胞降解活性明显优于阳性对照-人源瘦素蛋白(H-LEP)和市售药物罗格列酮(Rosiglitazone,Ros)，其降脂活性具有明显的浓度依赖性和细胞分化时期的依赖性，在脂肪细胞分化前期(Preadipocytes)、分化期(D0-D2)和成熟脂肪细胞期(D9)三个时期的细胞裂解作用显著优于两个阳性对照(H-LEP或罗格列酮)，特别CD环和E螺旋区突变的瘦素活性肽的降脂活性在D0-D2期，浓度为10-6M效果最优，从而为研制新型、高效、廉价的减肥和降血糖药物提供候选靶标。
附图说明：
图1是CD环和E螺旋区突变的瘦素活性肽的设计.A:以人源Leptin序列为模板模拟构建蛋白质三维结构图；B:人源Leptin3号外显子(上)与CD环和E螺旋区突变的瘦素活性肽(下)的氨基酸序列比对及功能分区.*：突变位点；
图2是CD环和E螺旋区突变的瘦素活性肽活性分析.3T3-L1前脂肪细胞降脂活性分析(MTT检测).PEP-E:CD环和E螺旋区突变的瘦素活性肽，其后的10-6、10-9、10-11分别指的是CD环和E螺旋区突变的瘦素活性肽的浓度为10-6M、10-9M、10-11M；H-LEP：人源瘦素蛋白(阳性对照1)，10-9是指10-9M；Ros：罗格列酮Rosiglitazone(阳性对照2)；Control：阴性对照。Preadipocytes：诱导分化前期；D0-D2：诱导分化第0-2天(分化初期)；D9：诱导分化第9天(成熟脂肪细胞期)。统计分析：a,阳性对照(H-LEP/Ros)vs.阴性对照(Control)，阳性对照的脂肪细胞裂解活性显著优于阴性对照P<0.05；b,PEP-E vs.H-LEP，PEP-E的脂肪细胞裂解活性显著优于H-LEP,P<0.05；c,PEP-E vs.Ros，PEP-E的脂肪细胞裂解活性显著优于Ros,P<0.05；*,P<0.01。
具体实施方式：
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
实施例1：
1.材料与方法
1.1 CD环和E螺旋区突变的瘦素活性肽的设计与合成
以人源Leptin氨基酸序列为模板，根据突变位点的亲/疏水性质变化情况在其功能区域设计一条CD环和E螺旋区突变的瘦素活性肽，其氨基酸序列为：SCHLHWAPGLETLDSGVQEASGY(具体如SEQ ID NO.1所示)。CD环和E螺旋区突变的瘦素活性肽交由上海生工生物工程有限公司合成，经HPLC检测，化学合成的CD环和E螺旋区突变的瘦素活性肽的浓度>95.23％，满足后续活性分析的要求。用质谱法检测合成CD环和E螺旋区突变的瘦素活性肽的准确性，确认其氨基酸序列为SCHLHWAPGLETLDSGVQEASGY(具体如SEQ IDNO.1所示)。
1.2瘦素活性肽的体外降脂活性分析
以3T3-L1前脂肪细胞系为模型，在细胞水平分析合成CD环和E螺旋区突变的瘦素活性肽的脂肪降解活性。具体操作如下：
1.2.1实验设计
(1)分组：
a:分化前期(Preadipocytes)；
b:分化初期D0-D2(诱导分化第0-2天)；
c:成熟脂肪细胞D9(诱导分化第9天)；
(2)瘦素多肽及对照
a.CD环和E螺旋区突变的瘦素活性肽(PEP-E)：用纯净水稀释成10-3M，-20℃保存备用。实验开始后，工作浓度为10-6M、10-9M、10-11M3个浓度梯度。
b.阳性对照1：H-LEP，重组人源瘦素(Leptin)蛋白(10221-HNAE，北京义翘神州生物技术有限公司)，用纯净水稀释成10-6M，-20℃保存备用。实验开始后，工作浓度为10-9M。
c.阳性对照2：Ros，罗格列酮(Rosiglitazone,R2408,Sigma)，用DMSO稀释成浓度为2518μM的贮存液，-20℃保存备用。实验开始后，用细胞培养液稀释成0.5μM的工作液。
d.阴性对照(Control)：等量的PBS
(3)细胞分化诱导液
a.细胞分化诱导液I：含有0.5μM IBMX(3-Isobutyl-1-methylxanthine,I5879,Sigma)，1μM地塞米松(dexamethasone,D4902,Sigma)，167nM胰岛素(insulin,I5500,Sigma)的完全培养基。
b.细胞分化诱导液II：含167nM胰岛素(insulin,I5500,Sigma)的完全培养基。
1.2.23T3-L1前脂肪细胞培养
(1)完全培养液：10％FBS(胎牛血清fetal calf serum)的DMEM培养基—DMEM45ml+FBS 5 ml+双抗0.5-0.8ml(DMEM,Hyclone SH30243.01B 500ml高糖，含L-谷氨酰胺，丙酮酸钠，Fetal Bovine Serum,Invitrogen 10091-148，Penicillin-Streptomycin,Invitrogen 15140-122100×)。
(2)传代：吸去旧液，用无钙镁PBS洗2遍，加入0.5ml/T250.25％胰酶，消化约2min(待细胞大部分漂起来时)，用完全培养液中止消化。不离心，按1：3直接接种于新的25cm2培养瓶(T25)。3～4h后换液，生长约70％时及时传代。
1.2.33T3-L1前脂肪细胞诱导分化为成熟脂肪细胞
(1)接种3T3-L1前脂肪细胞于corning cell bind 96孔板中(共接种了3个板，分别为a板、b板和c板)，接种密度为4500个细胞/孔，做好标记，置于37℃、5％CO2培养箱中静置培养。每板每个样品设3个重复，整个实验重复3次。
(2)待细胞汇合至100％后，继续培养48小时(使细胞退出生长周期，开始诱导分化)。
(3)待细胞退出生长周期后，
a.分化前期：
ⅰ.拿出a板，分别加入不同量的CD环和E螺旋区突变的瘦素活性肽(PEP-E)使其工作浓度分别为10-6M、10-9M、10-11M3个浓度梯度、2个阳性对照，阴性对照(PBS)及设置空白对照(每个样品设3个重复孔)，置于37℃、5％CO2培养箱中继续静置培养。
ⅱ.培养至45小时时(即药物处理结束前3小时)，取出a板，测MTT(分化前期)。具体操作：每孔加入5mg/mL的MTT溶液20μL，继续置于37℃、5％CO2培养箱中静置培养3小时，吸弃孔中培养液，每孔加入150μL DMSO，水平振荡10min，酶标仪检测492nm处的吸光值，绘制生长曲线。
b.分化初期(D0-D2)：
ⅰ.拿出b板加入分化诱导液Ⅰ(D0)，然后分别加入CD环和E螺旋区突变的瘦素活性肽(PEP-E)、2个阳性对照，阴性对照(PBS)及设置空白对照(每个样品设3个重复孔)，置于5％CO2、37℃培养箱中静置培养。
ⅱ.培养至45小时时(即药物处理结束前3小时)，取出b板，测MTT(脂肪细胞分化初期，D0-D2)。具体操作：每孔加入5mg/mL的MTT溶液20μL，继续置于37℃、5％CO2培养箱中静置培养3小时，吸弃孔中培养液，每孔加入150μL DMSO，水平振荡10min，酶标仪检测492nm处的吸光值，绘制生长曲线。
c.成熟脂肪细胞期(D9)：
ⅰ.拿出c板每孔加入分化诱导液Ⅰ(D0)，继续置于37℃、5％CO2培养箱中静置培养48小时。
ⅱ.分化诱导液Ⅰ处理48小时(D2)后，拿出c板每孔加入分化诱导液Ⅱ，并继续置于37℃、5％CO2培养箱中静置培养。
ⅲ.分化诱导液Ⅱ处理48小时(D4)后，拿出c板换成正常的完全培养基，并继续置于37℃、5％CO2培养箱中静置培养。
ⅵ.换成正常完全培养基培养48小时(D6)后，拿出c板换液，并继续置于37℃、5％CO2培养箱中静置培养24小时。
ⅴ.培养至D7(第7天)，拿出c板换液(完全培养基)，然后分别加入CD环和E螺旋区突变的瘦素活性肽(PEP-E)、2个阳性对照，阴性对照(PBS)及设置空白对照(每个样品设3个重复孔)，置于5％CO2、37℃培养箱中静置继续培养48小时。
ⅳ.药物处理45小时时(即药物处理结束前3小时)，取出c板，测MTT(成熟脂肪细胞期，D9)。具体操作：每孔加入5mg/mL的MTT溶液20μL，继续置于37℃、5％CO2培养箱中静置培养3小时，吸弃孔中培养液，每孔加入150μL DMSO，水平振荡10min，酶标仪检测492nm处的吸光值，绘制生长曲线。
1.2.4统计分析
MTT检测数据用SPSS 17.0软件进行单因素方差分析(ANOVA)。
2.结果
2.1 CD环和E螺旋区突变的瘦素活性肽的设计与合成
我们设计了一条CD环和E螺旋区突变的瘦素活性肽，与人源Leptin相比有3个氨基酸突变位点，多肽序列如下：SCHLHWAPGLETLDSGVQEASGY(分子量为：2457.66，详见图1)。经HPLC检测，化学合成的CD环和E螺旋区突变的瘦素活性肽的浓度>95.23％，满足后续活性分析的要求。
2.2 MTT降脂活性分析
细胞活性分析结果表明，合成CD环和E螺旋区突变的瘦素活性肽(PEP-E)的脂肪细胞降解活性在3个细胞分化时期(前脂肪细胞期、分化初期和成熟脂肪细胞期)都明显优于阳性对照-人源瘦素蛋白(H-LEP)和市售药物罗格列酮(Rosiglitazone,Ros)，并且CD环和E螺旋区突变的瘦素活性肽的降脂活性在D0-D2期，浓度为10-6M效果最优(图2)。
Claims (4)
1.一种CD环和E螺旋区突变的瘦素活性肽，其特征在于，其氨基酸序列如SEQ ID NO.1所示。
2.一种编码权利要求1所述的CD环和E螺旋区突变的瘦素活性肽的编码基因。
3.权利要求1所述的CD环和E螺旋区突变的瘦素活性肽在制备减肥药物中的应用。
4.根据权利要求3所述的应用，其特征在于，所述的减肥药物为降解脂肪细胞药物。
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